A Turn-On Fluorescent Probe for Highly Selective Detection and Visualization of Hydrogen Sulfide in Fungi

Hydrogen sulfide (H2S) is a significant actor in the virulence and pathogenicity of fungi. The analysis of endogenous H2S in fungi benefits the prevention and treatment of pathogenic infections. Herein, a H2S-activated turn-on fluorescent probe named DDX-DNP was developed for the sensitive and selective detection of H2S. Using DDX-DNP, the ability of several oral fungi strains to produce H2S was identified, which was also validated using a typical chromogenic medium. In addition, DDX-DNP was successfully used for the visual sensing of endogenous H2S in fungal cells via microscope, flow cytometry, and colony imaging, along with a specific validation with the co-incubation of H2S production inhibitors in living cells. Above all, DDX-DNP could be used for H2S detection, the fluorescent imaging of fungi, and even the identification of related fungi.


Introduction
Hydrogen sulfide (H 2 S) was the third important signaling gas molecule discovered in the body, after carbon monoxide (CO) and nitric oxide (NO), and it has an unpleasant smell of rotten eggs [1,2].Generally, H 2 S can be produced by enzymatic or non-enzymatic reactions.Now, several main enzymes related to the production of H 2 S have been characterized in mammals and bacteria, such as cystathionine-γ-lyase (CSE), cystathionineβ-synthase (CBS), cysteine aminotransferase (CAT), and 3-mercaptopyruvate sulfurtransferase (3-MST) [3][4][5][6].CSE and CBS produce H 2 S through the condensation of homocysteine and cysteine or the α,β-elimination of cysteine, while 3-MST reductively converts 3-mercaptopyruvate to H 2 S [2,4,5].In particular, in Candida albicans, CYS4 is annotated as encoding CBS for the synthesis of H 2 S in the Candida Genome Database [4].On the other hand, polysulfides and glutathione could be converted to H 2 S via the non-enzymatic pathway [6].As a well-known endogenous gas molecule, H 2 S has been known to exert all kinds of biological functions, such as relaxing vascular smooth muscles, mediating neuronal transmission, and regulating inflammation [7][8][9].However, an abnormal level of H 2 S in biosystems could induce an array of malignant diseases including Down Syndrome, Alzheimer's disease, and diabetes, as well as accelerate the proliferation and migration of cancer cells [10][11][12][13][14].In addition, H 2 S, a volatile sulfur compound, is also one of the main causes of oral malodor [15].Excessive H 2 S may also irritate the eyes and respiratory tract, resulting in the severe loss of consciousness, respiratory failure, and even death [16].
Given the biological importance of H 2 S, in the past few years, several methods have been reported for H 2 S detection and analysis, such as colorimetry, electrochemical assay, and gas chromatography [17][18][19].Unfortunately, few of these techniques are suitable for living systems or for the in situ monitoring and analysis of H 2 S. On the contrary, compared with the traditional methods, small molecular fluorescent probes have attracted considerable attention because of their inherent advantages, for example, excellent selectivity, high sensitivity, real-time monitoring, nondestructive visualization, convenience, and so on [20][21][22][23][24][25].
Some fluorescent probes have been developed for the detection of H 2 S based on a variety of strategies, such as (1) azides and nitro compounds being reduced to amines, (2) the Michael addition reaction of the H 2 S-specific cleavage of the disulfide bond, nucleophilic reaction, and thiolysis reaction, and (3) the H 2 S-induced metal displacement approach and the metal indicator displacement approach [26][27][28][29][30][31].These probes can image H 2 S in living cells, tissue, and blood samples in vivo, even in subcellular targeting location analysis [32][33][34][35].Although great advancements have been made, unfortunately, none of them has been used in microorganisms, including for fungi detection and analysis.Hence, the design and synthesis of a highly selective fluorescent probe for monitoring H 2 S in fungi is of great importance and urgently desirable to better understand the detailed physiological and pathological effects of H 2 S.
In view of this, we designed and synthesized a fluorescent probe named DDX-DNP for the detection of H 2 S in fungi based on the thiolysis of the dinitrophenyl ether strategy [31,36,37].Further research shows that the probe DDX-DNP has excellent optical performance, including biological applicability and good selectivity.More importantly, this probe successfully performed the visual sensing of endogenous H 2 S in fungi.To the best of our knowledge, this technique is a pioneering and effective tool for assessing H 2 S changes in the field of pathogenic infections.

Design of DDX-DNP and H 2 S Response
To achieve the detection and imaging of H 2 S, in consideration of its dual nucleophilicity toward H 2 S, we introduced the 2,4-dinitrophenyl (DNP) group, which not only acts as a recognition site but also an effective fluorescence quencher [36].The final structure of the designed fluorescent probe is shown in Scheme 1.In the absence of H 2 S, the probe DDX-DNP exhibited a weak fluorescence signal.When DDX-DNP reacted with H 2 S, the DNP group was removed from DDX-DNP so as to release the DDX-OH, exhibiting a remarkable fluorescence signal at 620 nm.Thus, H 2 S was detected qualitatively by recording the fluorescence intensity changes from probe DDX-DNP to the fluorophore, DDX-OH.
Some fluorescent probes have been developed for the detection of H2S based on a variety of strategies, such as (1) azides and nitro compounds being reduced to amines, (2) the Michael addition reaction of the H2S-specific cleavage of the disulfide bond, nucleophilic reaction, and thiolysis reaction, and (3) the H2S-induced metal displacement approach and the metal indicator displacement approach [26][27][28][29][30][31].These probes can image H2S in living cells, tissue, and blood samples in vivo, even in subcellular targeting location analysis [32][33][34][35].Although great advancements have been made, unfortunately, none of them has been used in microorganisms, including for fungi detection and analysis.Hence, the design and synthesis of a highly selective fluorescent probe for monitoring H2S in fungi is of great importance and urgently desirable to better understand the detailed physiological and pathological effects of H2S.
In view of this, we designed and synthesized a fluorescent probe named DDX-DNP for the detection of H2S in fungi based on the thiolysis of the dinitrophenyl ether strategy [31,36,37].Further research shows that the probe DDX-DNP has excellent optical performance, including biological applicability and good selectivity.More importantly, this probe successfully performed the visual sensing of endogenous H2S in fungi.To the best of our knowledge, this technique is a pioneering and effective tool for assessing H2S changes in the field of pathogenic infections.

Design of DDX-DNP and H2S Response
To achieve the detection and imaging of H2S, in consideration of its dual nucleophilicity toward H2S, we introduced the 2,4-dinitrophenyl (DNP) group, which not only acts as a recognition site but also an effective fluorescence quencher [36].The final structure of the designed fluorescent probe is shown in Scheme 1.In the absence of H2S, the probe DDX-DNP exhibited a weak fluorescence signal.When DDX-DNP reacted with H2S, the DNP group was removed from DDX-DNP so as to release the DDX-OH, exhibiting a remarkable fluorescence signal at 620 nm.Thus, H2S was detected qualitatively by recording the fluorescence intensity changes from probe DDX-DNP to the fluorophore, DDX-OH.

Spectral Characteristics and DFT Calculations of DDX-DNP and DDX-OH
In order to evaluate the detection properties of DDX-DNP, first, the optical spectral characteristics of DDX-DNP and the product, DDX-OH, were recorded using UV-vis absorbance and fluorescence spectroscopy.As shown in Figure 1a, DDX-DNP and DDX-OH displayed the maximum absorbance at 540 nm and 580 nm, respectively.Excited by laser at 580 nm, DDX-OH showed a strong fluorescence intensity at 620 nm, while DDX-DNP showed no fluorescence signal (Figure 1b).In addition, we also recorded the absorbance spectra of DDX-DNP at different concentrations (0-10 µM), and a good linear relationship was observed between the DDX-DNP concentrations and the absorbance values (Figure S1).
ton.In contrast, on the LUMO level, these electrons were mainly distributed in the DNP group.This phenomenon means that a possible photoinduced electron transfer (PET) process occurs in DDX-DNP; the electron transfer from the dihydroben-zo[c]xanthen skeleton to the DNP group leads to a fluorescence "turn off" state.Nevertheless, the π electrons of the DDX-OH fluorophore were mainly delocalized in the whole-molecule skeleton on both the LUMO and HOMO levels, which resulted in a typical intramolecular charge transfer (ICT) process, and the strong fluorescence signal was "turned on".In addition, the calculated energy gaps (LUMO → HOMO) of DDX-DNP and DDX-OH were 2.19 eV and 2.73 eV, respectively.In order to gain a better insight into the fluorescence response behavior of DDX-DNP from the perspective of the molecular orbital, the geometries of DDX-DNP and DDX-OH were optimized, and their frontier relevant molecular energy levels were calculated as shown in Figure 1c [36,38].The π electrons on the HOMO level of fluorescent probe DDX-DNP were mainly concentrated on the dihydrobenzo[c]xanthen skeleton.In contrast, on the LUMO level, these electrons were mainly distributed in the DNP group.This phenomenon means that a possible photoinduced electron transfer (PET) process occurs in DDX-DNP; the electron transfer from the dihydroben-zo[c]xanthen skeleton to the DNP group leads to a fluorescence "turn off" state.Nevertheless, the π electrons of the DDX-OH fluorophore were mainly delocalized in the whole-molecule skeleton on both the LUMO and HOMO levels, which resulted in a typical intramolecular charge transfer (ICT) process, and the strong fluorescence signal was "turned on".In addition, the calculated energy gaps (LUMO → HOMO) of DDX-DNP and DDX-OH were 2.19 eV and 2.73 eV, respectively.
Next, the fluorescence response of DDX-DNP toward H 2 S at different concentrations (0-100 µM) was also investigated.As can be seen in Figure 2a, along with the addition of incremental amounts of H 2 S, the fluorescence intensity enhancement at 620 nm, in a concentration-dependent manner, was observed clearly, which indicated that the DNP moiety was cut off by H 2 S to induce a dramatic and rapid fluorescence turn-on response.A point worth emphasizing is that a good linear relationship (R 2 = 0.9937) was observed between the H 2 S concentrations and the fluorescence intensity at 620 nm (Figure 2b).Furthermore, we measured the visual fluorescence images with a series of concentrations for H 2 S (0-100 µM) and DDX-OH (0-1 µM) in 96 plates, which showed that the fluorescence intensity gradually increased with the increasing H 2 S concentration (Figure 2c,d), indicating the potential visual detection of H 2 S. Above all, DDX-DNP could be activated by H 2 S, suggesting its application as a turn-on fluorescent probe for H 2 S.
tions (0-100 µM) was also investigated.As can be seen in Figure 2a, along with the add tion of incremental amounts of H2S, the fluorescence intensity enhancement at 620 nm, a concentration-dependent manner, was observed clearly, which indicated that the DN moiety was cut off by H2S to induce a dramatic and rapid fluorescence turn-on respons A point worth emphasizing is that a good linear relationship (R 2 = 0.9937) was observ between the H2S concentrations and the fluorescence intensity at 620 nm (Figure 2 Furthermore, we measured the visual fluorescence images with a series of concentratio for H2S (0-100 µM) and DDX-OH (0-1 µM) in 96 plates, which showed that the fluore cence intensity gradually increased with the increasing H2S concentration (Figure 2c,d indicating the potential visual detection of H2S.Above all, DDX-DNP could be activat by H2S, suggesting its application as a turn-on fluorescent probe for H2S.On the other hand, the fluorescence response of DDX-DNP toward H2S wi different incubation times, in the range of 0-180 min, was also explored.With the i crease in incubation time, the fluorescence intensity at 620 nm showed an increasi tendency, and it appeared to be flat after 120 min (Figure 3).Therefore, we finally ide tified 100 µM of H2S and an incubation time of 60 min as the key factors for the H2S fl orescence detection system based on the above results.In addition, kinetic analysis (Fi ure S3) revealed that the apparent rate constant k′ for the reaction of DDX-DNP wi H2S was 0.023 min −1 , and the pseudo-first-order rate constant k for the reaction DDX-DNP with H2S was 0.23 M −1 min −1 .
Job's Plot [39] was generated by continuously varying the mole fraction of NaH from 0 to 1 in a solution of NaHS + DDX-DNP with a total concentration of 100 µM.T fluorescence intensity became the strongest when the molar fraction of NaHS was 0 which indicated that the binding stoichiometry between probe DDX-DNP and NaHS 1:1 (Figure S4).On the other hand, the fluorescence response of DDX-DNP toward H 2 S with different incubation times, in the range of 0-180 min, was also explored.With the increase in incubation time, the fluorescence intensity at 620 nm showed an increasing tendency, and it appeared to be flat after 120 min (Figure 3).Therefore, we finally identified 100 µM of H 2 S and an incubation time of 60 min as the key factors for the H 2 S fluorescence detection system based on the above results.In addition, kinetic analysis (Figure S3) revealed that the apparent rate constant k ′ for the reaction of DDX-DNP with H 2 S was 0.023 min −1 , and the pseudo-firstorder rate constant k for the reaction of DDX-DNP with H 2 S was 0.23 M −1 min −1 .
Job's Plot [39] was generated by continuously varying the mole fraction of NaHS from 0 to 1 in a solution of NaHS + DDX-DNP with a total concentration of 100 µM.The fluorescence intensity became the strongest when the molar fraction of NaHS was 0.5, which indicated that the binding stoichiometry between probe DDX-DNP and NaHS is 1:1 (Figure S4).

Selectivity of DDX-DNP for the Detection of H2S
In this study, the fluorescence intensities of DDX-DNP and DDX-OH under different pH conditions were determined and analyzed.As shown in Figure 4a, DDX-DNP showed almost no variation from pH 2.0 to 12.0, and DDX-OH with the hydroxyl group exposed to the outside manifested a strong fluorescence signal from pH 6.0 to 9.0.
In order to compare the reaction rates at different pH values and confirm whether the probe could function smoothly under the studied physiological conditions, the fluorescence intensity of probe DDX-DNP toward H2S was investigated at different pH values (Figure 4b).The probe DDX-DNP's fluorescence intensity was enhanced noticeably from pH 4 to 9, and it reached the maximum fluorescent intensity at pH 7, while the other pH values did not exhibit any obvious fluorescence enhancement.In fact, fluorescence quenching occurred under strong alkali conditions.Therefore, pH 7.4 was determined to be the optimal pH for the incubation system.The incubation temperature was also measured, as shown in Figure S5.The reaction rate reached its maximum at 37 °C, and it did not continue to accelerate with the increasing temperature.

Confirmation about the Reaction between DDX-DNP and H2S
In order to confirm the sensing mechanism of DDX-DNP for H2S, HPLC-combined electrospray ionization-mass spectrometry (ESI-MS) analysis experiments were performed.As illustrated in Figure S11, after incubation with NaHS, a new ion peak at m/z 320.40 was observed, which could be assigned to the product DDX-OH [M − H] − .There- In order to compare the reaction rates at different pH values and confirm whether the probe could function smoothly under the studied physiological conditions, the fluorescence intensity of probe DDX-DNP toward H 2 S was investigated at different pH values (Figure 4b).The probe DDX-DNP's fluorescence intensity was enhanced noticeably from pH 4 to 9, and it reached the maximum fluorescent intensity at pH 7, while the other pH values did not exhibit any obvious fluorescence enhancement.In fact, fluorescence quenching occurred under strong alkali conditions.Therefore, pH 7.4 was determined to be the optimal pH for the incubation system.The incubation temperature was also measured, as shown in Figure S5.The reaction rate reached its maximum at 37 • C, and it did not continue to accelerate with the increasing temperature.
The excellent selectivity of the fluorescent probe for the target substance is an essential condition for its practical application.Thereby, we further explored the selectivity of DDX-DNP toward H 2 S. As shown in Figure 4c, in the presence of various substances such as common metal ions, Na + , K + , Mn 2+ , Ca 2+ , Cu 2+ , Zn 2+ , Fe 2+ , Mg 2+ , Ba 2+ , Ni 2+ , Cr 3+ , Fe 3+ , IO 4 − , SO 4 2− , CO 3 2− , and Cr 2 O 7 2− , and natural amino acids, Glu, Lys, Tyr, Gly, His, Arg, Ser, Trp, and biothiols (Cys, Met, GSH), the fluorescence intensity of DDX-DNP was measured.Notably, only glutathione and cysteine could trigger a weak fluorescence signal enhancement and cause hardly any interference in H 2 S detection.Other species could not render an apparent change in fluorescence intensity, which demonstrated that DDX-DNP displayed a good selectivity for the detection of endogenous H 2 S.

Confirmation about the Reaction between DDX-DNP and H 2 S
In order to confirm the sensing mechanism of DDX-DNP for H 2 S, HPLC-combined electrospray ionization-mass spectrometry (ESI-MS) analysis experiments were performed.As illustrated in Figure S11, after incubation with NaHS, a new ion peak at m/z 320.40 was observed, which could be assigned to the product DDX-OH [M − H] − .Therefore, the reaction mechanism of DDX-DNP for sensing H 2 S is proposed, as shown in Scheme S2.
Moreover, the chromatograms were also recorded using LC-MS/MS data for the fluorescent probe DDX-DNP, product DDX-OH, and reaction solution.As shown in Figure S6, a chromatographic peak of the probe DDX-DNP at the retention time of 3.44 min was observed, and DDX-DNP displayed a narrow peak at 3.29 min.Upon the addition of NaHS, a peak at 3.44 min (DDX-DNP) along with a peak at 3.29 min (DDX-OH) was recorded.Altogether, these results convey that the H 2 S-triggered thiolysis of dinitrophenyl ether occurred, releasing the target fluorophore.

High-Throughput Screening for Fungi-Produced H 2 S from Tongue-Coating Fungi Strains Based on DDX-DNP
As it is well known, redox homeostasis is essential for host colonization by pathogens, while H 2 S has antioxidative properties and a protective bacterial effect against antibiotics [4], thus further aggravating the pathogenicity of pathogens.H 2 S is not only generated by bacteria, but also it has been previously reported that some fungi can produce H 2 S. In our previous work, we leveraged the cultivation and sequencing of tongue-coating samples from healthy individuals to create a massive fungi bank.Thus, we created sufficient and convenient fungi strains for the high-throughput screening of fungi-produced H 2 S based on DDX-DNP.As displayed in Figure 5a,b, eight yeast strains were identified with high yields of H 2 S production from tongue-coating fungi, among which the Candida xestobii and Wickerhamiella spandovensis strains were the most active H 2 S-producing strains.Candida albicans, as a pathogen, was also previously discovered to be a H 2 S-producing fungus [4].
thiol-containing compounds (Figure S7).Therefore, this assay acts as a specific indicator to evaluate the H2S production of fungi.As we expected, when the identified eight yeast stains were grown in the BiGGY agar, the brownish-black color of the colonies appeared (Figure 5c), which was consistent with the results identified using DDX-DNP, further indicating that the developed fluorescent probe was feasible for detecting H2S.

Fluorescence Imaging of Fungi via H2S Monitoring
Inspired by its satisfying sensing performance and good selective ability, the sensing of endogenous H2S in fungi by DDX-DNP was performed with validation using 2-(Aminooxy) acetic acid (AOA) as the inhibitor of H2S production in cells.Then, a fungi imaging experiment was carried out using Candida albicans.As shown in Figure 6b,  In addition, we used the BiGGY (bismuth sulfite glucose glycine yeast) agar plate to verify the production of H 2 S by these identified yeasts.Before the formal experiments, first, we measured the selectivity of the BiGGY medium toward H 2 S. In the medium, bismuth ammonium citrate could be reduced by the H 2 S donor NaHS to yield a brownish-black color, while failing to react with glutathione, methionine, cysteine, and other thiol-containing compounds (Figure S7).Therefore, this assay acts as a specific indicator to evaluate the H 2 S production of fungi.As we expected, when the identified eight yeast stains were grown in the BiGGY agar, the brownish-black color of the colonies appeared (Figure 5c), which was consistent with the results identified using DDX-DNP, further indicating that the developed fluorescent probe was feasible for detecting H 2 S.

Fluorescence Imaging of Fungi via H 2 S Monitoring
Inspired by its satisfying sensing performance and good selective ability, the sensing of endogenous H 2 S in fungi by DDX-DNP was performed with validation using 2-(Aminooxy) acetic acid (AOA) as the inhibitor of H 2 S production in cells.Then, a fungi imaging experiment was carried out using Candida albicans.As shown in Figure 6b, a nonfluorescence signal was observed in the blank group, and a strong fluorescence signal was detected for fungal cells after the co-incubation of DDX-DNP, which indicated the abundant generation of endogenous H 2 S in Candida albicans.Furthermore, in the presence of AOA, the Candida albicans cells co-incubated with DDX-DNP, showing weak fluorescence intensity, which validated the specific detection of endogenous H 2 S by DDX-DNP.Subsequently, the ability of the fluorescent probe DDX-DNP to detect endogenous H 2 S in Candida albicans was also verified using a flow cytometric assay.As shown in Figure 6c, compared with the blank group (blue line), Candida albicans incubated with DDX-DNP displayed a significant shift (orange line); however, the shift in the inhibitor group (red line) was clearly reduced.At the same time, we also selected a fungus, W. paraugosa, which released almost no H 2 S as a negative strain for fluorescence imaging experiments.There is no doubt that no fluorescence intensity was observed in its fluorescence field (Figure 6d), which was in accordance with the results obtained in the fungi solution detection (Figure 5b).This further demonstrates the highly selective characteristic of endogenous H 2 S sensing using DDX-DNP.Additionally, the fluorescence imaging of Candida albicans and other yeast strains was also determined using agar plates.The fungal colonies displayed a significant fluorescence signal after being incubated with DDX-DNP, and the signal was measured using the Typhoon imager (Figure 6e,f).Thus, DDX-DNP has good biocompatibility and can achieve the detection and visualization of endogenous H 2 S created by living fungi, which suggests its potential applications for fungi identification or activity assays regarding H 2 S production.dogenous H2S in Candida albicans was also verified using a flow cytometric assay.As shown in Figure 6c, compared with the blank group (blue line), Candida albicans incubated with DDX-DNP displayed a significant shift (orange line); however, the shift in the inhibitor group (red line) was clearly reduced.At the same time, we also selected a fungus, W. paraugosa, which released almost no H2S as a negative strain for fluorescence imaging experiments.There is no doubt that no fluorescence intensity was observed in its fluorescence field (Figure 6d), which was in accordance with the results obtained in the fungi solution detection (Figure 5b).This further demonstrates the highly selective characteristic of endogenous H2S sensing using DDX-DNP.Additionally, the fluorescence imaging of Candida albicans and other yeast strains was also determined using agar plates.The fungal colonies displayed a significant fluorescence signal after being incubated with DDX-DNP, and the signal was measured using the Typhoon imager (Figure 6e,f).Thus, DDX-DNP has good biocompatibility and can achieve the detection and visualization of endogenous H2S created by living fungi, which suggests its potential applications for fungi identification or activity assays regarding H2S production.calculations were performed using Mos tool in GaussView 6.0.16, through a fchk-type file.The calculation was not limited by bonds/angles/dihedral angles, and all atoms could be freely optimized.Next, the HPLC elution conditions were optimized as follows: 0-1 min: 90% A; 1-2.5 min: 90-0% A; 2.5-4 min: 0% A; 4.0-5.5 min: 0-90% A; and 5.5-6.5 min: 90% A. The flow rate and the column temperature were set to 0.3 mL/min and 40 • C, respectively.Subsequently, the solutions of DDX-DNP and DDX-OH and the reacted solution of DDX-DNP and NaHS were injected, respectively.Finally, all the data were collected and processed using Analyst 1.6.3software.

Spectroscopic Measurements
Unless otherwise noted, all measurements were performed in KH 2 PO 4 -K 2 HPO 4 buffer (50 mM, pH 7.4).H 2 S was prepared from NaHS, which is a common donor and a standard source for H 2 S. The stock solution of the probe DDX-DNP (25 mM) was prepared in DMSO and then diluted to the desired concentration prior to the next experiment.The fluorescence spectra of the resultant solution were recorded using the maximum absorbance wavelength of the corresponding fluorophores as the excited wavelength (DDX-OH: λ ex = 580 nm).
For the effect of pH on the fluorescence probe, different pH values of the phosphate buffer from 2.0 to 12.0 were adjusted by supplementing appropriate volumes of standard phosphoric acid or potassium hydroxide, which were used for the fluorescence intensity detection of DDX-DNP.

Kinetic Studies [38]
The reaction of DDX-DNP (10 µM) with H 2 S in KH 2 PO 4 -K 2 HPO 4 buffer (50 mM, pH 7.4) at 37 • C was monitored by measuring the fluorescence intensity (E).The apparent rate constant for the reaction was determined by fitting the fluorescence intensity of the samples to the pseudo-first-order equation: ln where E t and E max are the fluorescent intensities at 620 nm at times t and the maximum value obtained during the reaction.k ′ is the apparent rate constant.The pseudo-first-order rate constant k (M −1 s −1 ) was obtained from Equation (2), where C is the concentration of H 2 S.

Fungi Strains and Cultivation
All the yeast strains from a single colony were initially propagated in liquid martin broth modified medium (MtB), supplemented with or without 2% agar, at 32 • C in an orbital shaker (130 rpm) or a constant-temperature incubator.The yeast strains studied in this work are listed in Table S1.The fugal cells were cultured to an optical density at 600 nm (OD 600 ) of 0.8 to 1.0, namely, culturing up to the logarithmic phase for experiments.

DDX-DNP High-Throughput Screening for H 2 S Production by Tongue-Coating Fungi
All the yeast strains were cultured in the MTB medium for at least 24 h to obtain enough fungal cells with an OD value of 1.0.Then, the probe DDX-DNP (25 µM) was added into the culture for co-incubation with yeast for about 4 h at 32 • C.After the fungal supernatants were harvested using centrifugation at 20,000× g for 10 min, the supernatant solutions were used to measure the fluorescence spectra using a microplate reader (λ ex 580/λ em 620 nm).

Fluorescence Imaging of Fungi via the Detection of H 2 S
The potential hydrogen sulfide-producing strains were cultured on MTB agar for 3-5 days.Fungal colonies were observed on the plates at 32 • C; then, the plates were divided into three regions, and DDX-DNP (25 µM) was sprayed on to the fungal colonies and incubated for 4 h at 32 • C. The inhibitor group were preincubated with AOA (1 mM) for 2 h at 32 • C. Finally, the plates were scanned using an Amersham Typhoon RGB (λ ex = 532 nm).
Candida albicans and negative strains were cultured in the MTB medium; then, DDX-DNP was added into the medium with a final concentration of 25 µM and incubated at 32 • C for 4 h.The inhibitor group were preincubated with AOA (1 mM) for 2 h at 32 • C.After washing the whole cells three times with PBS, they were dropped into glass slides and immobilized with coverslips for imaging experiments.Finally, the imaging cells were observed using a Leica Confocal Microscope with λ ex 561/λ em 602-650 nm.The fluorescent signal was also recorded using flow cytometry.A total of about 10,000 events were collected for data analysis, without using specific gating policies.The data were analyzed using FlowJoversion v10.8.1 software.

Conclusions
To sum up, we designed and synthesized a turn-on fluorescent probe for monitoring H 2 S based on the H 2 S-triggered thiolysis of the dinitrophenyl ether strategy.In the presence of H 2 S, the fluorescent probe DDX-DNP exhibits a remarkable fluorescence enhancement at 620 nm.More importantly, DDX-DNP was the first fluorescent probe successfully used for the fluorescence bioimaging of H 2 S in fungi.In short, DDX-DNP, as a turn-on fluorescent probe activated by H 2 S, can be used for H 2 S detection, as well as for the visual sensing of endogenous H 2 S in living fungi.

Figure 5 .
Figure 5. (a) The heat map of high-throughput screening of H2S-producing strains of tongue-coating fungi generated using DDX-DNP (25 μM); (b) the quantization results of 8 potential yeast strains with high yields of H2S; and (c) the visual detection of H2S generation of 8 yeast strains using BiGGY agar plates.

Figure 5 .
Figure 5. (a) The heat map of high-throughput screening of H 2 S-producing strains of tongue-coating fungi generated using DDX-DNP (25 µM); (b) the quantization results of 8 potential yeast strains with high yields of H 2 S; and (c) the visual detection of H 2 S generation of 8 yeast strains using BiGGY agar plates.

Figure 6 .Figure 6 .
Figure 6.(a) Chemical structure of the CBS inhibitor AOA; (b) fluorescence images of C. albicans incubated with DDX-DNP (25 µM) and in the presence of inhibitor AOA, scale bar = 25 µm; (c) Figure 6.(a) Chemical structure of the CBS inhibitor AOA; (b) fluorescence images of C. albicans incubated with DDX-DNP (25 µM) and in the presence of inhibitor AOA, scale bar = 25 µm; (c) flow cytometric analysis of C. albicans (blue) with DDX-DNP (orange) or both AOA and DDX-DNP (red); (d) fluorescence images of W. paraugosa incubated with DDX-DNP, scale bar = 20 µm; and (e,f) fluorescence images of yeast colonies on MtB agar plates along with bright fields.The plate was divided into three sections: (1) yeast colonies as the blank section in absence of DDX-DNP; (2) yeast colonies incubated with 25 µM DDX-DNP; and (3) yeast colonies incubated with 1 mM AOA and 25 µM DDX-DNP.